A multi-level mass spectrometry pipeline for the analysis of whole proteoforms and their complexes

蛋白质组学是一个非常宝贵的工具，用于阐明基础的发病的分子机制， 疾病蛋白质组的现代观点包括“蛋白质形式”的广泛复杂性，或 蛋白形式，由特定的遗传和化学修饰产生，可以改变和调节 蛋白生物活性在生物医学研究中，在三个不同的水平上分析蛋白质型是至关重要的：首先， 一级结构的确定，包括修饰的身份和位置;第二，数量 或这些修饰的丰度;以及最后，蛋白形式组装的表征。这些 蛋白质复合物通过弱相互作用结合在一起，是实际的生物活性分子 在体内运作的机器。目前，蛋白质型只能在全球范围内使用 质谱法（MS）。这一新兴领域被称为自上而下的蛋白质组学（TDP）， 成为下一代测序甚至基于肽的鸟枪水平上的可扩展解决方案 蛋白质组学，由于需要先进的技术来精确测量大蛋白质。为此 由于这个原因，TDP研究通常限于<30 kDa的蛋白形式，其代表了不到一半的蛋白形式。 哺乳动物蛋白质组此外，这些研究最常在还原、变性、 促进预分析工作流程的条件，因此蛋白质型的高阶结构丢失 以及半胱氨酸连接的翻译后修饰（PTM）。幸运的是，当代 结构生物学家可以在“类天然”条件下通过MS分析多蛋白复合物（MPC）， 保留非共价相互作用的条件，尽管该技术与高分子相互作用不相容。 通量研究可以用来发现新的生物学见解。在这里，我们提出一个新的 TDP管道，扩展了在发现模式下可测量的蛋白质组的质量范围， 整合变性和天然MS以测量MPC。我们首先将联合收割机创新分离 技术和气相化学（例如，新的离子碎裂技术和离子-离子反应） 改进高达100 kDa的蛋白质型的检测和测序，并允许表征 被忽视的PTM然后，将这些技术应用于>30 kDa的蛋白质组的定量 无论是在大规模的发现研究中（以确定健康和 疾病状态）和靶向实验（以监测感兴趣的蛋白质型的子集）。最后 通过变性TDP收集的单一蛋白形式的定性和定量信息 实验将被用来促进高通量天然MPC的自动化表征， MS研究。这种多层次的，以蛋白质组为中心的蛋白质组全球分析方法将提供 研究人员以其他方式无法获得的信息，开辟了新的可能性，制定新的 用于医学治疗和用于发现诊断疾病生物标志物的假说。

Proteomics is an invaluable tool for elucidating the molecular mechanisms that underpin the onset of disease. The modern vision of the proteome encompasses the broad complexity of “protein forms”, or proteoforms, created by specific sets of genetic and chemical modifications that can alter and regulate protein bioactivity. In biomedical research it is pivotal to analyze proteoforms at three distinct levels: first, determination of primary structure, including identity and position of modifications; second, the quantity or abundance of these modifications; and finally, characterization of proteoform assemblies. These protein complexes are held together by weak interactions and are the actual bioactive molecular machines that function in vivo. Currently, proteoforms can only be characterized at the global scale using mass spectrometry (MS). Known as top-down proteomics (TDP), this nascent field has not yet developed into a scalable solution on the level of next generation sequencing or even peptide-based shotgun proteomics, due to the advanced technology required to precisely measure large proteoforms. For this reason, TDP studies are typically limited to proteoforms <30 kDa, which represent less than half of the mammalian proteome. Furthermore, these studies are most often conducted under reducing, denaturing conditions to facilitate preanalytical workflows, so that higher order structure of proteoforms is lost together with cysteine-linked post-translational modifications (PTMs). Fortunately, contemporary structural biologists can analyze multi-proteoform complexes (MPCs) by MS under “native-like” conditions that preserve non-covalent interactions, although this technology is incompatible with high- throughput studies that can be leveraged to discover novel biological insights. Here, we propose a new TDP pipeline that extends the mass range of proteoforms measurable in discovery mode, and also integrates denaturing and native MS to measure MPCs. We will first combine innovative separation techniques and gas-phase chemistries (e.g., novel ion fragmentation techniques and ion-ion reactions) to improve the detection and sequencing of proteoforms up to 100 kDa, and allow the characterization of neglected PTMs. Then, these technologies will be applied to the quantification of proteoforms >30 kDa both in large-scale discovery studies (to identify proteoform expression variations between healthy and disease states) and in targeted experiments (to monitor subsets of proteoforms of interest). Finally, the qualitative and quantitative information collected on single proteoforms through denaturing TDP experiments will be used to facilitate the automatized characterization of MPCs in high-throughput native MS studies. This multi-level, proteoform-centric approach to the global analysis of proteomes will provide researchers with otherwise inaccessible information, opening new possibilities to formulate novel hypotheses for medical treatment and for the discovery of diagnostic disease biomarkers.